Fiber optic bio-sensor

ABSTRACT

A sensor for sensing and monitoring a property associated with transformation of a biochemical analyte by a micro-organism, the sensor comprising a glass permeable coating applied to an unclad portion ( 14 ) of a fibre optic member ( 13 ). The coating has a transformable precursor impregnated into it, the precursor being specifically metabolisable by one or more targeted organisms. When the sensor is placed in contact with a sample in a container ( 15 ) contacting an active targeted micro-organisms, the precursor is transformed by the micro-organisms to produce a spectroscopically detectable indicator of the property of the analyte. Spectroscopic information may be analysed by a computer program to provide an overall index of microbiological activity for the targeted micro-organism. The invention extends to a method of producing a sensor and a method of identifying the presence of a targeted micro-organism.

FIELD OF THE INVENTION

This invention relates generally to devices and methods for identifyingthe presence, and monitoring the activity, of a particularmicroorganism, including bacteria, in a sample. More specifically, thisinvention is associated with a device and methods based on the use ofoptical fibers applied to the detection, identification, and monitoringof a particular microorganism or species or group of microorganisms in asample including an environmental-, industrial- or human-derived sample.

BACKGROUND OF THE INVENTION

The isolation and identification of one or more microorganisms presentin a sample has long been of great importance in a wide range ofsituations. The fields in which the need for such procedures may ariseinclude medical diagnosis and treatment, military applications (e.g.biological warfare), security agencies, agriculture, food processing andwater quality assessment and control. In the field of medical diagnosisand treatment of both people and animals, time may be of the essence indetecting and characterising a microorganism in order to facilitateappropriate chemotherapeutic intervention. There are many otherinstances where the application of a rapid and relatively cheap test fordetermining the presence of microorganisms may be of great assistance.One example of such a situation arises in the case of Streptococcusmutans in saliva which is a predisposing factor to the development ofdental caries. Early identification and treatment may lead to asignificant decrease in tooth and gum disease.

Traditionally microorganisms such as bacteria have been identified bymethods which include cell culture, microscopy and more recentlyimmunoassay and nucleic acid probes. Culturing bacteria requires thedeposition of a sample onto a suitable culture medium such as an agarplate. The combination of sample and medium is incubated in a suitableenvironment and after a period of time such as 24-48 hours, colonies maybe harvested and subjected to identifying tests. If mixed colonies ofbacteria are grown it may be necessary to resample the growth and repeatthe process to obtain separate colonies of individual bacteria.

Identification has typically required subjecting the cells from thecultured colony to one or more characterising tests. Thesemicrobiological techniques often require optimum specimen quality toensure an accurate analytical result. These current techniques aremostly only qualitative, and are therefore difficult for monitoringbacterial activity quantitatively.

Bacterial structure may be a significant consideration in choosingappropriate tests. Cell walls of bacteria may comprise threemorphologically defined layers. The outermost layer may consist oflipids, polysaccharides and proteins. This layer distinguishes gramnegative bacteria from gram positive bacteria. The gram positivebacteria lack this outermost layer thereby providing the basis for oneof the most fundamental tests in microboiology.

Development of identification methods has been established from pastmicrobiological studies identifying specific biochemical reactions thatsome species or groups of microorganisms display. These specificmicroorganism mediated biochemical reactions form the basis oftraditionally practised laboratory methods for identifying andcharacterising species and strains.

With evolving technology, more sophisticated procedures such asfluorescent techniques and nucleic acid identification have also beenestablished. There has always been a great interest in rapididentification of disease related microorganisms. Towards this end,researchers have applied optical techniques such as Fourier TransferInfrared (FTIR) spectroscopy and fluorescence-based techniques. Theformer technique works on the principle of obtaining complex fingerprints from bacterial strains based on constituents of the cell wall.The latter technique typically employs fluorescing protein markers tomonitor bacterial activity. The FTIR techniques rely on algorithms andspectral analysis that compare corrugated spectroscopic patterns toidentify bacterial strains. This method requires the use of dryspecimens (after controlled heating in an oven) for analysis.Consequently, this technique is complex, time consuming and cannot beused directly on a patient. A fluorescence based technique is veryspecific for a bacterial reaction but requires an extended preparatoryand characterisation phase before the application of the sensor to theidentification of a bacterial strain. Application of these methods maybe difficult, expensive, time consuming and therefore not well adaptedfor routine use.

U.S. Pat. No. 5,496,700 to Ligler et al describes an optical immunoassayfor microbial analytes using non-specific dyes. Microorganisms in asample are all stained using a non-specific dye. The stained sample isplaced in contact with an optical wave guide which is coated with acapture molecule. A sample suspected of containing a microbial analyteis mixed with a dye and then exposed to the solid support material whichhas an attached capture molecule specific for the suspected microbialanalyte. In the preferred embodiment of the invention, the dye isfluorescent. Fluorescent emission technology utilises the ability ofsome compounds to absorb light of a particular wave length and emitlight of a different specific wave length. The use of such methodsusually requires considerable preparation time and cost in production.

Ligler's method relies on the fixation of a capture molecule to the waveguide device. In use a positive test will result from formation of acomplex including a dye, the microbial analyte and the capture molecule.This test therefore necessitates an initial dying step and theproduction of an analyte specific capture molecule which must be placedon the wave guide. The steps are relatively complex and sophisticatedand require the foundation step of producing an analyte specific capturemolecule and fixing it to the wave guide. The method and device isunlikely to be of particular use in vivo.

U.S. Pat. No. 6,256,522 to Schultz discloses a sensor for continuousmonitoring of biochemicals and a related method. The disclosure relatesto a sensor capsule having a processing chamber defined by a wall whichallows the passage of an analyte. Material capable of interacting withthe analyte is contained in the chamber. A light source, which may be anoptical fibre, causes light to impinge on a translucent portion of thechamber. Responsive fluorescent light is generated and emitted and maybe processed to determine concentration of an analyte. The disclosuredoes not describe an easy and effective method for determining thepresence of a microorganism such as a type of bacteria, in a sample andrelates to a relatively complex, sophisticated and costly piece ofequipment.

It would be advantageous to provide a quick and effective method ofdetermining the presence of, and monitor, microorganisms such asbacteria and preferably identifying the group, species or type ofmicroorganism.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in any country.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

In one form, although it need not be the only or indeed the broadestform, the invention resides in a sensor for sensing at least oneproperty associated with transformation of a biochemical analyte by amicroorganism, said sensor comprising:

at least one fibre optic member having at least one unclad portion;

a coating applied to the at least one unclad portion;

a precursor associated with the coating, said precursor transformable byat least one microorganism;

wherein

transformation of the precursor produces a spectroscopically detectableindicator of the at least one property.

The microorganism may be a prokaryote or eukaryote. A eukaryote includesa mammalian cell including a human cell, or other animal cell such as aninsect cell, yeast, fungus or amoebic cell. A prokaryote is particularlypreferred and includes all genera and/or species of bacteria.

The unclad portion of the fibre optic member is preferably a decladportion. The fibre optic member may have a plurality of unclad portions.The plurality of unclad portions may be contiguous, spaced or acombination of the two. The sensor may comprise two or more separatefibre optic members. The separate optic fibres may be substantiallyparallel.

Preferably the at least one fibre optic member has a first end adaptedto receive light from a light source. The at least one fibre opticmember may have a second outlet end adapted to co-operate with analysismeans to determine the presence of the spectroscopically detectableindicator. The at least one fibre optic member may be formed in a “Y”shaped configuration including three ends, a first end adapted toreceive light from a light source, a second end adapted to co-operatewith analysis means to determine the presence of the spectroscopicallydetectable indicator and a reflective end for reflecting light, thereflective end located on the equivalent and the declad portion of thelower most arm of the “Y” shape.

The coating is preferably a glass film. Suitably the glass film is bothporous and thin. The precursor may be immobilised within the coating.Alternatively or additionally the precursor may be immobilised on asurface of the coating. The precursor may comprise one or more ofD-mannitol, carbol fuchsine, methylene blue, sucrose or other suitablecompound.

The precursor may be selected to identify the presence of a singlemicroorganism species or, perhaps, variety. Alternatively, the precursormay be selected to identify two or more microorganism species orvarieties or a group thereof. Most preferably the precursor is selectedto identify one or more bacteria.

Transformation of the precursor may produce the spectroscopicallydetectable indicator directly. Alternatively, transformation of theprecursor may result in a product which cooperates with one or moreadjunctive compounds to produce the spectroscopically detectableindicator.

“Spectroscopically detectable” may include optically detectable. Thespectroscopically detectable indicator is preferably substantiallyformed in a zone of evanescent light waves adjacent to an outer surfaceof a fibre optic core of the at least one unclad region. Thespectroscopically detectable indicator may, in operation, be illuminatedby evanescent light waves.

In a second aspect the invention resides in a sensor system for sensingat least one property associated with transformation of a precursor byone or more microorganisms, said system comprising:

a fibre optic member having at least one unclad portion of optic fibre;

a coating applied to the at least one unclad portion;

a precursor associated with the coating, said precursor transformable byat least one microorganism; and

a light source adapted to co-operate with a first end of the fibre opticmember;

monitoring means adapted to co-operate with the unclad portion to detectan indicator signal;

wherein

transformation of the precursor by the one or more microorganismsproduces the indicator signal.

In a third aspect the invention resides in a method of producing asensor, said method comprising the steps of:

decladding one or more sections of a core of a fibre optic member;

applying a coating to the one or more sections, said coatingimmobilising a precursor to a spectroscopically detectable indicator,the precursor transformable to the detectable indicator by the activityof one or more microorganisms.

In a fourth aspect the invention resides in a method of identifying thepresence of at least one type of microorganism, the method comprisingthe steps of:

activating a light source in co-operative relationship to a first end ofa sensor as herein described;

monitoring the electromagnetic out-put from a coated, unclad section;

locating the sensor with its coated, unclad section in contact with asample; and

analysing the electromagnetic output to determine the presence of the atleast one type of microorganism.

Monitoring the electromagnetic output may comprise spectroscopicallymonitoring the electromagnetic output. The electromagnetic output may belight output.

Preferably the electromagnetic output is monitored through a second endof the sensor.

Locating the sensor may include immersing the sensor in a liquid sample.Locating the sensor may alternatively or further include placing theunclad section in contact with living tissue.

Analysing the electromagnetic output preferably comprises absorptionanalysis to identify the wave length of peak absorption ofelectromagnetic output.

Analysing the electromagnetic output may also include operating aprogrammable device programmed to receive digital information from aspectroscope and provide an analysis of results.

The light source may be any suitable apparatus and may comprise atungsten-halogen lamp. A xenon-arc lamp may also be used.

Preferably the monitoring means includes spectroscopic analysis means.The spectroscopic analysis means may include a processing systemincluding at least:

-   -   a) an input for receiving input data from a spectroscope;    -   b) a store for storing identification data for one or more        organisms; and    -   c) a processor, the processor being adapted to:        -   1) compare the input data to the identification data; and        -   2) generate a report indicating presence and type of one or            more microorganisms.

The processor may be programmed to store sample identification datawhich may include information such as sample origin, time and date ofcollection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic representation of a first embodiment of asensor of the present invention.

FIG. 2 shows a schematic representation of a second embodiment of asensor of the present invention.

FIG. 3 shows a side schematic view of a sensor probe.

FIG. 4A shows an example of a spectrum of light production from a lightsource.

FIG. 4B represents the evanescent wave distribution at the core-claddinginterface.

FIG. 4C represents an example of a transmission spectrum from a sensorsystem.

FIG. 5 shows a graph for light absorption over time for a sensor of thepresent invention.

FIG. 6 shows a graph for variation in intensity of the absorbance valleywhen using a sensor system of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a fibre optic microorganism sensorwhich may be used to detect and monitor one or more specific activitiesof microorganisms.

The term “sample” includes inter alia a biological, industrial and/orenvironmental sample. The term “biological sample” is used in itsbroadest sense and includes a sample of tissue or cells from a tissue ororgan isolated by, for example, surgical intervention, biopsy, lymphfluid, exudate (eg. pus, discharge), waste products (eg. urine, faeces),blood collection procedures or invasive or passive collectionprocedures. The expression “blood collection procedures” encompassserum, plasma and blood fractions. Furthermore, a biological sample maycomprise cells maintained in vitro culture or suspension. A biologicalsample is, therefore, a collection or population of cells which maycomprise a single cell type or comprise a mixed population of two ormore cell types. An environmental sample includes an industrial sampleand encompasses any location such as a water supply, food-handlingareas, terrestrial locations, waste-dumps, commercial areas etc.

As contemplated herein microorganisms include prokaryotic and eukaryoticcells. Prokaryotic cells include any bacterial or microbial cell such aspresent in an environmental or biological sample. Such prokaryoticorganisms include Pseudomonas sp., E. coli, Enterobacter sp., Salmonellasp., Klebsiella sp., Acetobacter sp., Porphroymonas sp., Staphylococcoussp., Streptococcus sp., Bacillus sp., Proteus sp., Helicobacter sp.,Campylobacter sp. or Legionella sp. amongst many others. Viruses includehepatitis virus, a retrovirus, an AIDS virus (e.g. HIV), foot and mouthdisease virus or polio virus amongst many others. Eukaroytic cellsinclude eukaryotic organisms such as yeast, fungi, amoeba, and othersingle cell organisms as well as cells from higher plants or animals.

Bacteria may include E. coli stains such as but not limited to, WA803,WA802, RR1, Q359, Q538, P2392, NM621, NM554, NM477, MC4100, MC1061,DL538, DB1316, CSH18, CES200, C600hfi, C600, BNN102, BNN93, BL21(DE3),and BHB2690. Other suitable bacteria may include but are not limited tothe following bacteria, Aminobacterium mobile DSM 12262, Aminomonaspaucivorans DSM 12260, Asaia bogorensis JCM 10569, Bacteroidesthetaiotaomicron BTX, Burkholderia kururiensis JCM 10599, Desulfovibriodechloracetivorans SF3, Escherichia coli HS(pFamp)R, Kocuria rhizophilaDSM 11926, Methylobacterium mesophilicum AM24, Mycobacterium avium MAC511, Mycobacterium avium MAC 101, Phormidium corium, Pseudomonasaeruginosa ERC1, Pseudomonas aeruginosa HER-1001, Pseudomonas aeruginosaHER-1002, Pseudomonas aeruginosa HER-1010, Pseudomonas aeruginosaHER-1009, Pseudomonas aeruginosa HER-1016, Pseudomonas aeruginosaHER-1017, Pseudoxanthomonas broegbemensis DSM 12573, Ralstonia gilardiiLMG 5886, Shewanella frigidimarina ACAM 591, Shewanella gelidimarinaACAM 456, Streptococcus pneumoniae MS22, Streptococcus pneumoniae Fi10,Streptococcus pneumoniae 51702, Streptococcus pneumoniae TW31,Streptococcus pneumoniae TW17, Thiomicrospira frisia JB-A2,Thiomicrospira kuenenii JB-A1, Treponema lecithinolyticum OMZ 685,Treponema maltophilum BR, Treponema maltophilum PNA1, Treponemamaltophilum H02A, Ureaplasma urealyticum. Still other microorganisms mayinclude but are not limited to the following fungal cells Hyphodontiaaustralis 231Kluyveromyces lactis CK56-7A, Kluyveromyces lactis CW64-1C,Prosthemium asterosporum A1, Prosthemium betulinum B1 Saccharomycescerevisiae 1A-H19 [psi-], Saccharomyces cerevisiae 5V-H19 [psi-],Saccharomyces cerevisiae 1-5V-H19, Saccharomyces cerevisiae PS-5V-H19,Saccharomyces cerevisiae C10B-H49, Saccharomyces cerevisiae 9V-H70[PIN+], Saccharomyces cerevisiae 4V-H73, Saccharomyces cerevisiae17G-H73, Saccharomyces cerevisiae 3B-H72, Saccharomyces cerevisiae DL1,Saccharomyces cerevisiae GW226, Saccharomyces cerevisiae JM43-GD7,Saccharomyces cerevisiae MCC318, Saccharomyces cerevisiae NB39-5D,Saccharomyces cerevisiae NGB108, Saccharomyces cerevisiae PTH43,Saccharomyces cerevisiae PTH352, Saccharomyces cerevisiae PTY11,Saccharomyces cerevisiae TF112, Saccharomyces cerevisiae TWM1041,Saccharomyces kluyveri GRY1175, Saccharomyces kluyveri MCC328 andSaccharomyces kluyveri NB180.

A eukaryotic organism includes a yeast, fungus, amoeba, parasite, insectand the like.

Preferably the utility of the present system arises from the capacityfor tailoring the system to one or more wide range of types ofmicroorganisms including bacteria and specific strains of bacteria, bythe selection of a relevant biochemical reagent or precursor forincorporation in the sensor region of the fibre optic system. Thisprovides a device which may perform measurements rapidly andqualitatively and even quantitatively. The system provides a safe meansto analyse biological materials and biochemical reactions.

The method of the present invention may be conveniently classed intothree phases being:

-   -   1) a fibre optic transduction phase;    -   2) a biochemical recognition phase; and    -   3) a spectroscopic analytical phase.

The fibre optic transduction phase arises from transmission of lightthrough an optical fibre during which total internal reflection takesplace at an interface between the core of the fibre optic member and anexternal cladding. During each total internal reflection, a certainportion of electromagnetic radiation or wave penetrates the cladding.This wave is called the evanescent wave. The present sensor utilises asection of fibre optic material which has been decladd or had thecladding removed. At this point, the exponentially decaying portion ofthe evanescent wave is harnessed to interact with the medium thatsurrounds it. In the sensor, the evanescent wave absorption phenomenonat the core cladding interface of an optical wave guide is used todetermine different physical and chemical variables associated withmicroorganisms, and particularly bacterial, activity.

Typically an optical fibre is formed by making a preformed glasscylinder, drawing the fibres from the preform and testing the fibres.The glass for the preform is usually made by a process called modifiedchemical vapour deposition (MCBD). In MCBD, oxygen is bubbled throughsolutions of silicon chloride (SiCL₄), germanium chloride (GeCl₄) and/orother chemicals. The precise mixture governs the various physical andoptical properties such as the index of refraction, coefficient ofexpansion and melting points. The gas vapours may then be conducted tothe inside of a synthetic silica or quartz tube which forms thecladding. Under the effect of heat, silicon and germanium react to formsilicon dioxide and germanium dioxide which deposit on the inside thetube and fuse together to form glass. A lathe is used to form an evencoating and a consistent diameter. Purity of the glass may be maintainedby using corrosion resistant plastic and in the gas delivery system, byprecisely controlling the flow and composition of the mixture. Thepreformed blank is allowed to cool and is then loaded into a fibredrawing tower.

The blank may be lowered into a gravity furnace, which melts the tipuntil a pendulous blob falls down with gravity and forms a thread. Thisthread is then passed through a series of coating cups and ultra violetcuring ovens to result in a product that has an inner core and outercladding and often a buffer outer coating. The cladding is outer opticalmaterial surrounding the core and designed to reflect light back intothe core. The buffer coating may be a plastic coating that protects thefibre from damage and moisture.

The fibres may be formed as single mode fibres which are used totransmit one signal per fibre or multi-mode fibres which may be used totransmit many signals per fibre. The single mode fibres have small coresof approximately 9 microns in diameter while multi-mode fibres havelarger cores which may be up to 62.5 microns in diameter.

Cladding may be removed in a number of ways. One preferred procedure isa chemical etching process carried out by immersing the preferred regionof the fibre in a 50% hydrofluoric acid solution for a period of 20 to30 minutes.

In the biochemical recognition phase, a biochemical reaction which isunique to a microorganism or group of microorganisms is selected toidentify the presence of the microorganisms. A precursor ortransformable element of this reaction is selected for localisation in acoating placed over the denuded or declad optic fibre. Transformation ofthe precursor by microorganisms may result in a spectroscopicallydetectable indicator, the presence of which will be monitored byappropriate analysis means. Additionally, or alternatively, a specificindicator may also be selected wherein the transformed precursor willactivate an adjunctive, co-operating indicator to provide a more easilydetectable reaction.

In applying a coating to the optical fibre it is preferable to use asol-gel technique. The sol-gel technique is utilised to form a porous,glass thin film coating around the cladding denuded optical fibre.Preparation of the sol-gel may be done at room temperature by thehydrolysis and condensation reaction of Tetra Ethyl Ortho Silica (TEOS)in an acidic environment, to form siloxane polymer, leading to gelation.The chemical reaction is as shown in the following equation:Si(OC₂H₅)₄+₄H₂O→Si(OH)₄+4C₂H₅OHSi(OH)₄→SiO2+2H₂O

The hydrolysis reaction of TEOS proceeds via the replacement of —OC₂H₅groups by OH groups. Nominally, four H₂O molecules are required for thecomplete hydrolysis of a Si—(OC₂H₅)₄ molecules to form Si—(OH)₄molecule.

The starting solution may be prepared by the partial hydrolysis of TEOSaccording to the above procedure. Denatured anhydrous ethanol, deionisedwater and hydrochloric acid may be used to perform the hydrolysis ofTEOS. The entire mix may be maintained under constant stirring for 1hour using a magnetic stirrer and then stored at room temperature. After24 hours, the precursor solution and optical indicator may be mixedthoroughly into the material. This prepared sol may be used to coat theunclad portion of the optical fibre. Dip coating equipment may be usedfor this purpose.

The outer sol-gel coating displays three-dimensional porosities. Thepreferred pore size ranges from 10 nm to 100 nm. However, the preferredpore size may change with different applications. The porosity can bevaried by: (1) altering the drying procedure of the sol-gel film, and(2) varying the molar ratios/chemical composition of the precursorchemicals.

In the spectroscopic analytical phase, absorption spectrometry may beused based on the fact that a given molecular species absorbs light in aspecific region of the spectrum, and in varying degrees, that absorptionpattern is characteristic of the particular species. In this system, theindicator or precursor is selected based on the products of metabolismor other chemical activity by microorganisms which display an absorptionspectrum that may serve as a finger print for bacterial identificationand monitoring purposes.

Spectroscopy is employed widely in laboratory diagnostics to studychemical processes such as metabolic reactions and enzyme kineticsamongst other things. Instruments reliant on spectroscopy make use ofthe absorption, emission or scattering of electromagnetic radiation inthe examination of atoms or molecules of interest. In doing so, rapidqualitative and quantitative study of molecules has become possiblehastening processes such as medical diagnostics and quality testing.

Briefly absorption spectroscopy utilises the transition between energylevels in molecules absorbing electromagnetic radiation. Ultraviolet andvisible light incite electrons in atoms and molecules to higher energylevels and the amount of light energy absorbed is a function of theincident light wavelength. The unique absorption spectra displayed bydifferent chemical species makes spectroscopy an indispensable tool inmodern diagnostics.

Intensity of any given absorbent spectrum has a linear relationship withthe concentration of the chemical species with that given absorptionspectra. Hence, the levels of any given analyte can be rapidlydetermined using modern spectroscopic instruments thereby providing aquantitative indication of the amount of the material present.

Referring to FIG. 1 there is seen a sensor system of the presentinvention generally designated as 10. The sensor system comprises alight source 11 which may conveniently provide light of a mixed and wideband length. The light is directed into an inlet end 12 of an opticalfibre 13. A tungsten-halogen lamp may be used. Use of a photosensitiveindicator in the present system, may result in output in the visiblerange. However, a xenon-arc lamp may also be used, especially whenhigher intensity and broader light spectrum is required. Other suitablelight sources may be used.

A declad section 14 of the optical fibre 13 is provided with a coating.The coating immobilises a transformable precursor for a specificmicroorganism mediated reaction chosen for application. The coating is abio-inert coating and may be preferably formed according to the abovedescription as a thin film polymer layer. The coating may be permeableto microorganisms under review to facilitate interaction. The decladregion 14 may be located in a specimen chamber 15 defined by an outerwall 16. The optical fibre 13 has a discharge end 17 for discharginglight. The discharge end is adapted to cooperate with a spectrometer 18which forms analysis means. In operation a sample such as saliva islocated in the specimen chamber 15. Light is provided from the lightsource 11. At the declad region 14 active targeted cells, if present,metabolise these selected precursors to produce an indicator colour orother indicium such as an absorption pattern. The reaction may producean indicator by activating a specific indicator which may be separatelyincluded in the outer coating as an adjunct to the transformableprecursor.

The sensor system may produce an immediate response which may bemonitored by a spectroscopic method in real time. The response, in somesituations, will increase with time.

This method of using evanescent wave spectroscopy in conjunction withmicroorganisms and, in particular bacteria induced and mediated reactionto monitor microbial activity in, for example, body fluids provides auseful and quick indication of the presence of bacteria. The presentsensor system process includes the sensor phase, the biochemicalrecognition phase and the signal transduction phase all in one opticalfibre. It displays a very short response time and high sensitivity whenused. This invention therefore introduces a combination of the benefitsof, for example, established bacterial mediated chemical reactionidentification and fibre optic spectroscopy. The invention produces asensor with high sensitivity and specificity associated with an abilityto rapidly identify bacteria and bacterial activity in real time.

A sensor of the present invention may be prepared as a probe for use invivo or alternatively may be provided for ex Vivo application. While thebelow examples and discussion are directed to bacteria it should beunderstood that the sensor method may be turned to other types ofmicroorganisms.

Referring to FIG. 2 there is seen a sensor system generally indicated as19 incorporating a light source 20. A lens 21 may be effectivelyharnessed to regulate light output and the angle of incident rays. Lightis delivered to a fibre optic member 22 which has a sensor element 23which is also shown in close detail. The sensor element comprises adeclad section 24 of a fibre optic core 25 and surface gel coating 26.The surface gel coating 26 is preferably formed as a bio-inert substancecontaining a selected biochemical precursor. The bacteria in a samplemay interact with the precursor to produce optically detectableindicators. The sensor element 23 may be located in a channel 27 in atest pad 28. The sensor element 23 may be secured to the floor of thechannel 27 using wedges so that a very small quantity of sample isrequired to adequately immerse the sensor portion. This embodiment ofthe device provides an easy accessible test region with a lowrequirement for quantity of specimen.

The detectable indicator may be optically detectable and produces lightwhich continues along the fibre optic member 22 to a spectrometer 29. Atthis point, an inquiry is made of presented light and data is inputthrough lead 30 to a processing means in the form of computer 31.Preferably the computer has a data store of information relating tocharacteristics of individual bacterial species or varieties. Thecomputer may be programmed to compare incoming data against the datastore to thereby provide an indication of the identity of bacteriapresent. Further the intensity and degree of the spectrometric resultsmay result in an estimation of the quantitative concentration ofbacteria in a sample.

Referring to FIG. 3 there is seen a probe 32 for a sensor system of thepresent invention comprising an inner fibre optic core 33 having anexternal cladding 34. The cladding is removed in a sensor zone 35 andreplaced by an external coating 36 of a bio-inert material whichincludes a preselected precursor for metabolism by bacteria of interest.The interrupted cladding continues 34 and the cladding 34 and inner core33 are terminated by a reflecting surface 37 which reflects incidentlight back up the fibre optic core for analysis. The probe isparticularly useful as it may be inserted into cavities of a patient orinto other samples which may be difficult to access or which may betoxic. The reflected light may be subsequently analysed according to theabove description.

The light source produces a transmission spectrum as shown in FIG. 4A(depending on the nature of light) wherein wavelength (λ) is plottedagainst intensity (T). The evanescent wave distribution at thecore-cladding interface can be represented as in FIG. 4B. It has amaximum intensity in proximity to the core and a taper in intensity awayfrom the core. When a photosensitive indicator is immobilized within aporous class coating at the cladding denuded optical fiber, subsequentlythe transmission spectrum obtained is as shown in FIG. 4C. The valley inthe transmission spectrum is due to the absorbance of the photosensitiveindicator at a specific wavelength.

The power transmission in an optical fiber, having an absorbingcladding, is given by the modified Beer-Lambert's law:P(I)=P ₀exp(λI)  (1)where I is the distance along the unclad portion of the fiber, P0 is thepower transmitted in the absence of an absorbing species and (is theevanescent wave absorption coefficient.The above equation can be rewritten as,P(I)=P ₀exp(rαI)  (2)r is the fraction of the power transmitted through the cladding and (isthe bulk absorption coefficient of the cladding. The evanescent waveabsorbance ‘A’ from the previous equations as log P0/P(I).$\begin{matrix}{A = {\frac{\gamma\quad l}{2.303} = \frac{r\quad\alpha\quad l}{2.303}}} & (3)\end{matrix}$

FIG. 5 shows a graph demonstrating another example of another example ofresults from the use of the biosensor. Two peaks are provided atproximity 480 and 640 nanometer wavelengths. Increasing results as shownbetween 0 minutes, 45 minutes and 60 minutes as plots 38, 39 and 40,respectively. The pattern may be specific for a transformed precursor orassociated indicator. Presence of this indicator may be conclusiveevidence of the present of live and active bacteria of a type underinvestigation.

FIG. 6 is a typical graph representing the activity profile ofstreptococcus mutans with sucrose in human saliva monitored by thepresent fibre optic sensor. This graph represents the increase in theformation of extra cellular polysacchride and lactic acid adjacent or onthe sensor from 5 minutes to 120 minutes time duration. The increase inby-product formation with time show two slopes. In the first phase, theslope is significantly smaller than the second phase. This denotesincreasing activity (greater concentration) of the by-product formationin the second phase compared to that of the first. The change wasdetected by the fibre optic sensor system of the present invention.

It is clear to the skilled addressee that multiple sensor sections maybe used on one single fibre optic member. Such sensor sections may becontiguous or alternatively may be separated by areas of clad centralcore. In a further alternative, multiple fibre optic members may beused. These multiple members may be arranged substantially parallel andmay produce a bank or array of sensors to provide multiple results.

EXAMPLE 1 Staphylococcus aureus

Staphylococcus Aureus is a pathogenic bacterium that causes significantmorbidity particularly to immune compromised individuals. It is also acommon food borne bacterium. Nosocomial infections due to this bacteriumcreate problems that are increasing in severity and are a financial andhealth liability in the clinical environment. It has become important todevelop a rapid detection system for Staphylococcus aureus and inparticular methicillin resistant Staphylococcus aureus.

A conventional growth and indication medium for the detection ofStaphylococcus aureus is mannitol salt agar, which is both a selectiveand differential growth medium. It is used to differentiate pathogenicStaphylococcus species from non-pathogenic members of the genusMicrococcus. The medium typically contains about 7.5% salt thusselecting for organisms that are able to tolerate the presence of highlevels of salt. This medium also contains an indicator, phenol red,which is a pinkish red at neutral pH, red at pH at 7.4 and above and isyellow below the pH 6.8. Organisms that ferment mannitol produce acid asa reaction product hence causing colour change to the indicator.

A sample containing Staphylococcus aureus was placed in contact with thesensor portion of the present invention. Production of acid fromD-mannitol in the presence of methicillin resulted in an opticalindicator, which was detected by spectroscopic means, providing adistinctive absorption spectrum.

EXAMPLE 2 Mycobacterium

The infectious agents of tuberculosis and leprosy belong to the samebacterial genus, Mycobacterium. Infection by Mycobacterium tuberculosiscauses fever, cough, loss of energy and weight loss and serious lungdamage. Leprosy, an infection of the skin, peripheral nerves and mucousmembranes is caused by Mycobacterium leprae. The serious nature of thesemycobacteria makes the need for a rapid diagnosis necessary to ensureappropriate therapeutic treatment can be initiated as early as possible.

A sample containing Mycobacterium leprae and Mycobacterium tuberculosiswas placed in contact with the sensor element of the present system.Carbol fuchsine was dispersed in the outer coating of the sensorelement. Reaction of carbol fuchsine with lipids of the mycobacteriumcell wall produced an absorption pattern which was distinctive andindicated the presence of the mycobacteria.

EXAMPLE 3 Environmental Sampling

General bacterial contamination in the environment may be identifiedusing the system of the present invention. Reaction with general dyessuch as methylene blue and features of bacteria such as the cell wallmay produce an optically or spectroscopically detectable indicator.

A contaminated sample with mixed bacterial population was placed incontact with the sensor element of the present invention. A generalabsorption pattern was detected by spectroscopic methods to indicate thepresence of multiple bacterial organisms.

EXAMPLE 4 Staphylococcus mutans

Staphylococcus mutans is a reliable indicator of a predisposition todental caries. The outer coating of the sensor element of the presentsystem was impregnated with sucrose. The sensor element was brought intocontact with a sample containing Staphylococcus mutans which resulted inmetabolism of sucrose to form latic acid and polysaccharides, therebyresulting in an optically detectable indicator.

In a further version of this example, bacitracin was also mixed with thesample to render the test more specific for Staphylococcus mutans.

In one embodiment the system may include data processing means. The dataprocessing means may assign numerical values to certain characteristicsof identified microorganisms and in particular bacteria. The assigningof numerical values enables data processing means to assess the statusof a sample such as a biological or environmental sample. Dataprocessing may result in a quantitative indicator of the status ofcontamination of a sample or alternatively may provide a generic result,such as “low” or “moderate” and “high” contamination levels.

The types of attributes which may be ascribed a numerical value includebacterial genus, bacterial species, bacterial variety, bacterialconcentration and rate of development of change of indicator.

The value ascribed to each feature may be referred to as an index value(I_(v)).

The sum of I_(v), ie. ΣI_(v), provides a contamination index of a sample(C_(I)) value and this enables an analytical approach to screening andidentifying contamination of samples. Clearly the process may be equallydirected to identifying the health or presence of commensal organismsand normal healthy flora in certain samples. The (I_(v)) index value foreach feature may be stored in a machine readable storage program, whichmay be capable of processing the data to provide a contamination valuefor a sample or group of samples.

Thus, in another aspect, the invention contemplates a computer programproduct for assessing the status of presence of microorganisms of asample or group of samples, said product comprising:

code that receives as input index values for at least two featuresassociated with microorganisms where in the features are selected from agroup including:

a) genus of microorganism;

b) species of microorganism;

c) variety of microorganisms;

d) concentration of microorganisms; and

e) speed of development of indicators.

a code that adds the index values to provide a sum corresponding to acontamination index for the sample; and

a computer readable medium that stores the code.

In a preferred embodiment, the computer program product comprises codethat assigns an index value for each feature of the microorganism orgroup of microorganisms.

In a related aspect, the invention extends to a computer for assessingthe likelihood of contamination of a sample compound or group of sampleswherein the computer comprises:

a machine readable data storage medium comprising a data storagematerial encoded with machine readable data, wherein said machinereadable data comprise index values for at least two features associatedwith microorganisms wherein the features are selected from:

a) genus of microorganism;

b) species of microorganism;

c) variety of microorganisms;

d) concentration of microorganisms; and

e) speed of development of indicators;

a working memory for storing instructions for processing the machinereadable data;

a central processing unit coupled to the working memory and to themachine readable data storage medium, for processing the machinereadable data to provide a sum of the index values corresponding to apotency value for the compounds; and

an output hardware coupled to the central processing unit for receivingthe contamination values.

A version of these embodiments may be represented in a figure whichshows a system including a computer (31, FIG. 2) comprising a centralprocessing unit (“CPU”), a working memory which may be, for example, RAM(Random Access Memory) or “Core” memory, mass storage memory such as oneor more disk drives or CD ROM drives, one or more cathode ray tubedisplay terminals, one or more keyboards, one or more input lines andone or more output lines all of which are interconnected by aconventional bi-directional system bus.

Input hardware may be coupled to the computer by input lines which maybe implemented in a variety of ways. For example, machine readable dataof this invention may be inputted by the use of a modem or modemsconnected by a telephone line or dedicated data line. Alternatively oradditionally, the input hardware may comprise a CD. Alternatively, ROMdrives or disk drives in conjunction with display terminals, keyboardsand keyboard may also be used as a input device. The output device,coupled to a computer by output lines, may similarly be implemented byconventional devices. Output hardware might also include a printer sothat hard copy output may be produced, or a disk drive to store systemoutput for later use.

In operation the CPU coordinates the use of the various input and outputdevices coordinates data accesses from our storage and accesses to andfrom working memory and determines the sequence of data processing. Anumber of programs may be used to process the machine readable data ofthis invention.

The computer is located in signal connection with a spectroscope forreceiving and analysing input to produce at least one indicator of themicroorganism status of a sample.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the disclosure.

1. A sensor for sensing and/or monitoring at least one propertyassociated with transformation of a biochemical analyte by at least onemicroorganism, said sensor comprising: at least one fibre optic memberhaving at least one unclad portion; a coating applied to the at leastone unclad portion; a precursor associated with the coating, saidprecursor transformable by the at least one microorganism; whereintransformation of the precursor produces a spectroscopically detectableindicator of the at least one property.
 2. The sensor of claim 1 whereinthe unclad portion of the fibre optic member is a declad portion.
 3. Thesensor of claim 1 comprising a plurality of unclad portions.
 4. Thesensor of claim 3 further comprising two or more separate fibre opticmembers.
 5. The sensor of claim 1 further adapted to cooperate withanalysis means for determining the presence of the spectroscopicallydetectable indicator.
 6. The sensor of any one of the proceeding claimswherein the coating is a glass film.
 7. The sensor of the proceedingclaim wherein the glass film is both porous and thin.
 8. The sensor ofany one of the proceeding claims wherein the precursor is immobilisedwithin the coating.
 9. The sensor of the proceeding claim wherein theprecursor comprises one or more of D-mannitol, carbol fuchsine,methylene blue, sucrose or other suitable compound.
 10. The sensor ofclaim 1 wherein transformation of the precursor results in a productwhich cooperates with an adjunctive compound to produce thespectroscopically detectable indicator.
 11. A sensor system for sensingat least one property associated with transformation of a precursor byone or more microorganisms, said sensor system comprising: a fibre opticmember having at least one unclad portion of optic fibre; a coatingapplied to the at least one unclad portion; a precursor associated withthe coating, said precursor transformable by at the one or moremicroorganisms; a light source adapted to cooperate with a first end ofthe fibre optic member to provide input light to the fibre optic member;and monitoring means adapted to cooperate with the unclad portion todetect an indicator signal in received light from the fibre opticmember, said indicator signal indicative of the at least one property;wherein transformation of the precursor by the one or moremicroorganisms produces the indicator signal by interaction with theinput light to produce the received light.
 12. The sensor system ofclaim 12 wherein interaction with the light is interactive with anevanescent wave form of the input light.
 13. A method of producing asensor, said method comprising the steps of: decladding one or moresections of a core of a fibre optic member; applying a coating to theone or more sections, said coating immobilising a precursor to aspectroscopically detectable indicator, the precursor transformable tothe detectable indicator by the activity of one or more microorganisms.14. A method of identifying the presence of at least one type ofmicroorganism comprising the steps of: activating a light source incooperating relationship to a first end of a sensor according to any oneof claims 1 to 11; monitoring the electromagnetic output from a coatedunclad section; locating the sensor with its coated unclad section incontact with the sample; and analysing the electromagnetic output todetermine the presence of the at least one type of microorganism. 15.The method of claim 14 wherein monitoring the electromagnetic outputcomprises spectroscopically monitoring the electromagnetic output. 16.The method of either one of claim 14 or claim 15 wherein analysing theelectromagnetic output comprises conducting absorption analysis toidentify wave lengths of peak absorption of electromagnetic output. 17.The method of claim 16 wherein analysis of the electromagnetic outputincludes operating a programmable device programmed to receive digitalinformation from a spectroscope and provide an analysis of results. 18.The method of claim 17 wherein the programmable device is programmed toidentify one or more features of the at least one microorganism, the oneor more features being selected from a group including genus ofmicroorganism, species of microorganism, variety of microorganism,concentration of microorganism and speed of development of indicator.19. The method of claim 18 wherein the programmable device is furtherprogrammed to ascribe an index value to each identified feature andprovide an overall index for a sample according to the algorithmC _(s) =ΣIv where: C=and overall index Iv=individual indices.
 20. Amethod of coating a sensor for sensing and/or monitor at least oneproperty associated with transformation of a biochemical analyte by atleast one microorganism, comprising steps of: making a coating mixtureby dispersing a precursor in a sol-gel solution; wherein the precursoris transformable by the at least one microorganism; and coating thesensor with the coating mixture; wherein the sensor comprises at leastone fiber optic member having at least one unclad portion, and thecoating is preferably applied to the unclad portion.
 21. The method ofcoating a sensor of claim 20, wherein the sol-gel solution is made byhydrolysis of Tetra Ethyl Ortho Silica; wherein the molecule used forhydrolysis is selected from the group consisting of H₂O, anhydrousethanol, and hydrochloric acid.
 22. The method of coating a sensor ofany of claims 20-21, wherein the sol-gel coating is done by dip coating.23. The method of coating a sensor of any of claims 20-22, wherein theprecursor is selected from the group consisting of D-mannitol, carbolfuchsine, methylene blue, and sucrose.
 24. The method of costing asensor of any claims 20-23, wherein the resultant product from thetransformable precursor cooperates with an adjunctive compound toproduce the spectroscopically detectable indicator.